Method for detecting Chlamydia trachomatis and kit therefor

ABSTRACT

A method for detecting  Chlamydia trachomatis  comprising performing PCR using DNA obtained from a sample as a template and detecting an amplification product, wherein a primer pair used for PCR is designed on the basis of the nucleotide sequences of the regions corresponding to the nucleotide numbers 5157 to 5201 and 5245 to 5276 in the nucleotide sequence of SEQ ID NO: 1 so that the nucleotide sequence between the two regions can be amplified. A method for quickly detecting  Chlamydia trachomatis  with superior sensitivity and specificity is provided.

RELATED APPLICATIONS

This is the U.S. National Phase under 35 U.S.C. § 371 of InternationalApplication PCT/JP2004/002435, filed Feb. 27, 2004, which was publishedin a language other than English, which claims priority of JP2003-050662, filed Feb. 27, 2003.

TECHNICAL FIELD

The present invention relates to a method for detecting Chlamydiatrachomatis and a kit therefore.

BACKGROUND ART

Chlamydia trachomatis is one of nongonococcal urethritis pathogens, andas a method of detecting it, a method of amplifying a partial sequenceof a cryptic plasmid existing in Chlamydia trachomatis cells in amultiple copy number by a gene amplification method and detecting theamplification product is known (Japanese Patent Nos. 2719225 and3127135).

DISCLOSURE OF THE INVENTION

An object of the present invention is to provide a method for quicklydetecting Chlamydia trachomatis with superior sensitivity andspecificity and a kit therefor.

The inventors of the present invention found that, if PCR was performedwith primers designed for a specific region in the cryptic plasmidpLGV440 of Chlamydia trachomatis, Chlamydia trachomatis could be quicklydetected, and thus accomplished the present invention.

The present invention provides a method for detecting Chlamydiatrachomatis comprising performing PCR using DNA obtained from a sampleas a template and detecting an amplification product, wherein a primerpair used for PCR is designed on the basis of the nucleotide sequencesof the regions corresponding to the nucleotide numbers 5157 to 5201 and5245 to 5276 in the nucleotide sequence of SEQ ID NO: 1 so that thenucleotide sequence between the two regions can be amplified (detectionmethod of the present invention).

The present invention also provides a kit used for the method of thepresent invention, that is, a kit for detection of Chlamydia trachomatisby performing PCR using DNA obtained from a sample as a template, whichcomprises a primer pair designed on the basis of the nucleotidesequences of the regions corresponding to the nucleotide numbers 5157 to5201 and 5245 to 5276 in the nucleotide sequence of SEQ ID NO: 1 so thatthe nucleotide sequence between the two regions can be amplified(detection kit of the present invention).

In the present invention, the primer pair preferably consists of anoligonucleotide having an nucleotide sequence of SEQ ID NO: 2, 3 or 4and an oligonucleotide having a nucleotide sequence of SEQ ID NO: 5 or6, more preferably an oligonucleotide having the nucleotide sequence ofSEQ ID NO: 3 and an oligonucleotide having the nucleotide sequence ofSEQ ID NO: 5, or an oligonucleotide having the nucleotide sequence ofSEQ ID NO: 4 and an oligonucleotide having the nucleotide sequence ofSEQ ID NO: 6.

The present invention also provides a hybridization probe containing anoligonucleotide designed on the basis of the nucleotide sequence of theregion corresponding to the nucleotide numbers 5210 to 5245 in thenucleotide sequence of SEQ ID NO: 1 and a label.

The nucleotide sequence of SEQ ID NO: 1 is the nucleotide sequence ofthe cryptic plasmid pLGV440 of the Chlamydia trachomatis (GenBankaccession number X06707), and those skilled in the art can easilyidentify and recognize the region specified by the nucleotide numbers ofthe nucleotide sequence of SEQ ID NO: 1 also in a strain having amutation in pLGV440 by taking difference in the nucleotide sequence thatmay exist depending on individuals or the like into consideration.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 shows a production time course of an amplification product(detection results of real-time PCR): a: no copy, b: 2 copies, c: 20copies, d: 200 copies, and e: 2000 copies.

BEST MODE FOR CARRYING OUT THE INVENTION

<1> Detection Method of the Present Invention

The detection method of the present invention is a method for detectingChlamydia trachomatis comprising performing PCR using DNA obtained froma sample as a template and detecting an amplification product, wherein aprimer pair used for PCR is designed on the basis of the nucleotidesequences of the regions corresponding to the nucleotide numbers 5157 to5201 and 5245 to 5276 in the nucleotide sequence of SEQ ID NO: 1 so thatthe nucleotide sequence between the two regions can be amplified.

The sample is not particularly limited so long as it contains orpossibly contains Chlamydia trachomatis. Examples thereof include urine,urethra swab, cervical swab and so forth. From these samples, DNA can beobtained by a usual method under conditions where DNA of the crypticplasmid of Chlamydia trachomatis can be prepared.

PCR in the detection method of the present invention can be performedaccording to a usual PCR procedure except that DNA obtained from thesample is used as a template, and that the specific primer pair is used.

The primer pair used in the present invention is designed on the basisof a nucleotide sequence of a region corresponding to the nucleotidenumbers 5157 to 5201 in the nucleotide sequence of SEQ ID NO: 1 (firstregion) and a nucleotide sequence of a region corresponding to thenucleotide numbers 5245 to 5276 (second region) so that the nucleotidesequence between the two regions can be amplified.

The length of the primers is usually 10 to 40 nucleotides. Further, theposition in each region and the length of the primers are preferably setso that the Tm value should become 55 to 70° C., and thus the annealingtemperature used in PCR can be set to be relatively high. The Tm valueused herein is a value calculated by the nearest neighbor base pairanalysis. The primers constituting the primer pair are preferablydesigned to have substantially the same Tm values.

Specific examples of the primer designed on the basis of the firstregion include a primer having the nucleotide sequence of SEQ ID NO: 2(corresponding to the nucleotide numbers 5157 to 5185 in the nucleotidesequence of SEQ ID NO: 1) or a nucleotide sequence complementarythereto, a primer having the nucleotide sequence of SEQ ID NO: 3(corresponding to the nucleotide numbers 5171 to 5201 in the nucleotidesequence of SEQ ID NO: 1) or a nucleotide sequence complementarythereto, and a primer having the nucleotide sequence of SEQ ID NO: 4(corresponding to the nucleotide numbers 5171 to 5196 in the nucleotidesequence of SEQ ID NO: 1) or a nucleotide sequence complementarythereto. Examples of the primer designed on the basis of the secondregion include a primer having the nucleotide sequence of SEQ ID NO: 5(corresponding to the nucleotide numbers 5276 to 5252 in the nucleotidesequence of SEQ ID NO: 1) or a nucleotide sequence complementarythereto, and a primer having the nucleotide sequence of SEQ ID NO: 6(corresponding to the nucleotide numbers 5276 to 5246 in the nucleotidesequence of SEQ ID NO: 1) or a nucleotide sequence complementarythereto.

The primer pair is designed so that the nucleotide sequence between thetwo regions can be amplified, that is, one primer should be a senseprimer and the other primer should be an antisense primer. Because thecryptic plasmid is a cyclic plasmid, it has two nucleotide sequencesbetween the two regions. However, the primers are usually designed sothat a shorter nucleotide sequence should be amplified.

Preferred examples of the primer pair include a combination of anoligonucleotide having a nucleotide sequence of SEQ ID NO: 2, 3 or 4 andan oligonucleotide having a nucleotide sequence of SEQ ID NO: 5 or 6.Further, more preferred examples of the primer pair include acombination of an oligonucleotide having the nucleotide sequence of SEQID NO: 3 and an oligonucleotide having the nucleotide sequence of SEQ IDNO: 5 and a combination of an oligonucleotide having the nucleotidesequence of SEQ ID NO: 4 and an oligonucleotide having the nucleotidesequence of SEQ ID NO: 6.

The primer pair for the aforementioned specific regions can be designedby a method known to those skilled in the art taking PCR conditions intoconsideration. The primer pair can be designed by using a computerprogram for designing primers.

The primers may be designed as a mixed primer obtained by mixing two ormore kinds of primers for one or both of the sense primer and theantisense primer. When a mutation of a nucleotide exists in the regionfor which the primer is designed, detection efficiency can be improvedby using a mixed primer.

Although the PCR conditions may be determined according to a usual PCRmethod, the annealing temperature should be set to be relatively highdue to the use of the aforementioned specific primer pair. The annealingtemperature is usually 50 to 70° C. Because the annealing temperature isrelatively high, the conditions for two-step PCR in which annealing andelongation are performed under the same conditions may be determined.

A typical example of composition of the PCR reaction mixture is asfollows.

DNA fragments 1 molecule or more Primers 100 to 2000 nM Nucleotides 100to 500 μM for each DNA polymerase 0.25 to 1.25 Units/μl Tris-HCl (pH 7to 9) 1 to 5 mM MgCl₂ 1.5 to 5 mM Surfactant or gelatin 0 to 25% (Finalvolume: 25 to 100 μl)

Further, a typical example of the temperature cycle is as follows, andsuch a temperature cycle is usually repeated 30 to 60 times.

-   (1) Denaturation: 90 to 95° C., 1 to 60 seconds-   (2) Annealing: 55 to 70° C., 6 to 60 seconds-   (3) Elongation: 72 to 75° C., 6 to 60 seconds

A typical example of the temperature cycle for the two-step PCR is asfollows, and this temperature cycle is usually repeated 30 to 60 times.

-   (1) Denaturation: 90 to 95° C., 1 to 60 seconds-   (2) Annealing and elongation: 55 to 70° C., 6 to 60 seconds

In the detection method of the present invention, the amplificationproduct can be detected by a usual method for detecting amplificationproducts. For example, the amplification product may be detected byagarose gel electrophoresis, or by real time PCR, in which PCR isperformed in the presence of a substance of which fluorescence changeswhen it binds to the amplification product (for example, a fluorescentdye of which fluorescence intensity changes when it binds to adouble-stranded DNA, a hybridization probe designed so that it shouldhybridize with a single-stranded DNA, and fluorescence intensity thereofshould change due to fluorescence resonance energy transfer (FRET) uponhybridization, and so forth), and then fluorescence is measured.

According to the detection method of the present invention, primershaving a relatively high Tm value can be designed for specified regions,and therefore the annealing temperature used in PCR can be maderelatively high. Accordingly, the time required to lower temperaturefrom the denaturation temperature to the annealing temperature isreduced. Further, non-specific amplification is reduced due to the highannealing temperature. Furthermore, two-step PCR in which annealing andelongation are performed under the same conditions is also possible. Asa result, quicker gene amplification is enabled.

The present invention also provides a hybridization probe suitable fordetection by hybridization. Examples of such a hybridization probeinclude a probe comprising an oligonucleotide designed on the basis of anucleotide sequence of a region corresponding to the nucleotide numbers5210 to 5245 in the nucleotide sequence of SEQ ID NO: 1 and a label.This oligonucleotide may be complementary to a sense chain orcomplementary to an antisense chain.

The chain length and Tm value of the oligonucleotide of thehybridization probe are suitably determined depending on thehybridization conditions. The length of this oligonucleotide is usually25 to 45 nucleotides, and the Tm value is usually 50 to 70° C. When thehybridization probe is used for real-time PCR, the Tm value of theoligonucleotide is preferably set to be higher than the Tm value of theprimer by 2 to 5° C. so that the probe should hybridize with a targetsequence before hybridization of the primer. Examples of such anoligonucleotide include an oligonucleotide having the nucleotidesequence of SEQ ID NO: 7.

The hybridization probe can be labeled in such a manner that thehybridization should not be inhibited under the hybridizationconditions. The hybridization probe for real-time PCR is preferablylabeled in such a manner that fluorescence intensity should change whenit hybridizes with a single-stranded DNA.

In the detection method of the present invention, the amplificationproduct is preferably detected by a detection method using thehybridization probe of the present invention.

<2> Detection Kit of the Present Invention

The detection kit of the present invention is a kit usable for thedetection method of the present invention, that is, a kit for detectionof Chlamydia trachomatis by performing PCR using DNA obtained from asample as a template, which is characterized by including a primer pairdesigned on the basis of the nucleotide sequences of the regionscorresponding to the nucleotide numbers 5157 to 5201 and 5245 to 5276 inthe nucleotide sequence of SEQ ID NO: 1 so that the nucleotide sequencebetween the two regions can be amplified.

The primer pair is as described above with regard to the detectionmethod of the present invention.

In the kit of the present invention, the primers of the primer pair maybe included as a mixture or included separately.

The kit of the present invention may further include reagents requiredfor PCR and/or detection of an amplification product in addition to theprimer pair. Examples of such reagents include the aforementionedhybridization probe.

EXAMPLES

Hereafter, the present invention will be explained more specificallywith reference to the following examples.

Example 1 Detection by PCR

From the first region (nucleotide numbers 5157 to 5201) and the secondregion (nucleotide number 5245 to 5276) of the nucleotide sequence ofthe cryptic plasmid of Chlamydia trachomatis (SEQ ID NO: 1, GenBankaccession number X06707), nucleotide sequences having a high GC content(48 to 55%) and high Tm value (63 to 65° C. (calculated by the nearestneighbor base pair method)) were selected. That is, 3 types of upstreamprimers (SEQ ID NOS: 2, 3 and 4) and 2 types of downstream primers (SEQID NOS: 5 and 6) were selected as target binding sequences. Then,oligonucleotides having these target binding sequences were synthesizedas primers.

TABLE 1 Upstream (forward) primers: CT-F5157-29: (SEQ ID NO:2) cag tcacac cca aaa gct ctg gga gca tg CT-F5171-31: (SEQ ID NO:3) agc tct gggagc atg ttc tta gtc tca gca g CT-F5171-26: (SEQ ID NO:4) agc tct ggg agcatg ttc tta gtc tc Downstream (reverse) primers: CT-R5276-25: (SEQ IDNO:5) tcg cgt agg gct tag aat cac ctt c CT-R5276-31: (SEQ ID NO:6) tcgcgt agg gct tag aat cac ctt ctc gta c

Three kinds of pairs each consisting of a combination of theaforementioned upstream and downstream amplification primers (SEQ IDNOS: 2-5, 3-5 and 4-6) were used for PCR. The reaction mixture (25 μL)contained 1×Gene Taq buffer, 0.625 U of Gene Taq polymerase (NipponGene), 0.2 mM each of dGTP, dCTP, dATP and dUTP, 0.5 μM each of theupstream and downstream primers, and 2 to 2000 copies of Chlamydiatrachomatis cell genomic DNA (ATCC strain CT-VR878). To examineappropriate reaction temperature, PCR was performed by using atemperature gradient for the annealing temperature for the amplificationreaction in iCycler (BIO-RAD). The reaction temperatures were asfollows: 95° C. for 4 minutes, (95° C. for 30 seconds, gradient of 62 to72° C. for 30 seconds, 72° C. for 30 seconds)×50 cycles, and 72° C. for7 minutes. Occurrence of amplification was confirmed by applying theproduct to 3% agarose gel electrophoresis.

Whichever primer pair was used, 2 copies of Chlamydia trachomatis cellgenomic DNA could be detected with a relatively high annealingtemperature condition of 64.0 to 68.2° C.

The above results showed that the aforementioned primer sequences hadsuperior sensitivity and specificity and were effective for reducing thereaction time.

Example 2 Detection Using Two-step PCR

Among the primer pairs used in Example 1, the primer pairs of SEQ IDNOS: 3-5 and 4-6 were used for two-step PCR. The reaction mixture (25μL) contained 1×ΔTth buffer, 0.625 U of ΔTth (TOYOBO), 0.2 mM each ofdGTP, dCTP, dATP and dUTP, 0.5 μM each of the upstream and downstreamprimers, and 2 to 2000 copies of Chlamydia trachomatis cell genomic DNA(ATCC strain CT-VR878). The reaction temperatures were as follows: 95°C. for 1 minute, (95° C. for 15 seconds, 65 or 68° C. for 20 seconds)×2cycles, and (90° C. for 15 seconds, 65 or 68° C. for 20 seconds)×58cycles. The reaction was performed by using a thermal cycler (TaKaRa).Occurrence of amplification was confirmed by applying the product to 3%agarose gel electrophoresis.

When the primer pair of SEQ ID NOS: 3-5 was used, 2 copies of Chlamydiatrachomatis cell genomic DNA could be detected with an annealingtemperature of 68° C. When the primer pair of SEQ ID NOS: 4-6 was used,2 copies of Chlamydia trachomatis cell genomic DNA could be detectedwith an annealing temperature of 65° C.

The above results showed that it was possible to perform two-step PCR inwhich the reaction time could be further reduced.

Example 3 Detection Using Real-time PCR

Among the primer pairs used in Example 1, the primer pair of SEQ ID NOS:3-5 was used for two-step PCR. The reaction mixture (25 μL) contained1×ΔTth buffer, 0.625 U of ΔTth (TOYOBO), 0.2 mM each of dGTP, dCTP, dATPand dUTP, 1 μM of the upstream primer, 0.5 μM of the downstream primer,0.2 μM of a probe for real-time detection (5FL-CT-5210-36: 5′-caa agctag aac aac gcc gcc ttc cat tct tga tgc-3′ (SEQ ID NO: 7), c at the 5′end was labeled with a fluorescent dye, type of the marker: BODIPY-FL(Molecular Probe)), and 2 to 2000 copies of Chlamydia trachomatis cellgenomic DNA (ATCC strain CT-VR878). The reaction temperatures were asfollows: 95° C. for 1 minute, (95° C. for 15 seconds, 65° C. for 20seconds)×2 cycles, (90° C. for 15 seconds, 65° C. for 20 seconds)×58cycles. The reaction was performed in iCycler (BIO-RAD). The real-timedetection procedure was according to Japanese Patent Laid-openPublication (Kokai) No. 2001-286300.

The results are shown in FIG. 1. With the primer pair used in thisexample, 2 copies of Chlamydia trachomatis cell genomic DNA could bedetected in real time with an annealing temperature of 65° C.

INDUSTRIAL APPLICABILITY

The present invention provides a method for quickly detecting Chlamydiatrachomatis with superior sensitivity and specificity.

1. A method for detecting Chlamydia trachomatis comprising performingPCR using DNA obtained from a sample as a template and detecting anamplification product, wherein a primer pair used for PCR consists of anoligonucleotide having the nucleotide sequence of SEQ ID NO: 2, 3 or 4and an oligonucleotide having the nucleotide sequence of SEQ ID NO: 5 or6, and wherein performing PCR comprises annealing at a temperature of 64to 68.2° C.
 2. The method according to claim 1, wherein the primer pairconsists of an oligonucleotide having the nucleotide sequence of SEQ IDNO: 3 and an oligonucleotide having the nucleotide sequence of SEQ IDNO: 5, or an oligonucleotide having the nucleotide sequence of SEQ IDNO: 4 and an oligonucleotide having the nucleotide sequence of SEQ IDNO: 6.